PROJECT SUMMARY It is clear that latently infected CD4+ T cells in cART treated HIV-infected individuals are the major reservoir and obstacle to eradication. This research proposal specifically intends to address the FOA entitled ?Silencing of HIV-1 Proviruses? with the following specific objective proposed by the FOA: ? to harness the PIWI RNA pathway to silence HIV?. In contrast to other small non-coding RNA silencing pathways (siRNA, shRNA, miRNA), the piRNA pathway has the theoretical advantage of permanently silencing genes through epigenetic modifications (H3K9 methylation) in addition to mRNA degradation activity, the latter being the primary mode of silencing by other small RNAs, thus, ensuring gene silencing in progeny cells. Our preliminary findings indicate that PIWI proteins are expressed in CD4+ T cells and T cell lines, and appear to be regulated by immune activation. It is unknown, however whether the PIWI-piRNA pathway can be induced to perform similar functions in somatic cells as they do in germ cells. We hypothesize that the piRNA-PIWI pathway is functional in primary CD4+ T-cells and can be manipulated to silence HIV. Attempts will be made to ensure success of the project by teaming experts in fundamental small noncoding RNA and piRNA biology with an HIV expert, all having long track records in their fields. In the R66 portion of the proposal we will test the proof of concept that that one can induce a piRNA pathway against HIV genes in ACH-2 cells, so that, when they are activated by cytokine, they have ceased to produce HIV virions. If successful, the R33 portion of the proposal will further characterize ACH-2 piRNAs that have activity against a broad range of HIV strains, including cross Clades within ex vivo primary cells taken from cART treated individuals. As part of pre-clinical development a CD4 receptor targeting chimeric aptamer-piRNA strategy will be explored. The current proposal will reveal further insights on the role of the piRNA pathway in human cells, whether they can be exploited as an HIV silencing strategy as part of the armamentarium toward HIV cure therapy.